Stroke is the third leading cause of death and the leading cause of disability in this country, affecting about 650,000 people in the US each year. Currently approved therapies for stroke are directed toward acutely restoring blood flow (using drugs that break up clot). A new approach is to use stem cells to regenerate portions of the brain that are damaged in a stroke. Stem cells can be obtained from adult individuals, or from embryos. Studies using adult stem cells have shown that only a small fraction of these cells are capable of transforming into brain cells. Another problem is that in patients with stroke, many different types of brain cells must be replaced. Furthermore, the replacement cells must reconstitute the normal architecture of the lost brain. Additionally, the stem cells must overcome the hostile metabolic milieu in the ischemic brain, which includes poor blood flow, as well as the adverse metabolic environment that caused the stroke in the first place (eg. high blood sugar, high cholesterol, high blood pressure).

So in this proposal we are taking a different approach. We will develop methods to make blood vessels using human embryonic stem cells (HESC). HESC derived blood vessel cells will be injected into rats that have had a surgically-induced stroke. We will determine if the HESC-derived vascular cells find their way to the area of poor blood flow. We will determine if these cells survive and if they generate new blood vessels.

In other works, rather than attempting to provide stem cells that will develop into complex brain tissue after stroke, we intend to first restore the brain vessels in the area of the stroke. We hypothesize that “if we build the road, they will come”, ie. the restoration of the brain vessels will enhance survival of brain tissue in areas of poor flow, and may induce repair of the injured area, by encouraging neighboring nerve cells to migrate into the area.

Furthermore, we plan to genetically engineer the HESCs to make them hardier. The area of stroke in the brain is a hostile environment for cells. One of the factors that mediates the adverse metabolic effects is a substance called ADMA (asymmetric dimethylarginine). We will engineer HESC that are more able to handle this substance, and determine if that genetic modification gives the HESC a better chance of surviving and forming blood vessels.

This proposal will provide insights into the use of human embryonic stem cells (HESC) for regenerating the injured brain after a stroke.

Statement of Benefit to California:

Every 45 seconds, someone in California has a stroke. Stroke is the third leading cause of death and the leading cause of disability in California. Each year, stroke kills more than twice as many Californian women as does breast cancer. Currently approved therapies for stroke are limited. The only FDA approved therapy that is directed toward stroke is thrombolysis (using drugs that break up clot). New approaches are needed.

A novel approach is to use stem cells to regenerate portions of the brain that are damaged in a stroke. Stem cells can be obtained from adult individuals, or from embryos. Studies using adult stem cells have shown that only a small fraction of these cells are capable of transforming into brain cells. Another problem is that in patients with stroke, many different types of brain cells must be replaced. Furthermore, the replacement cells must reconstitute the normal architecture of the lost brain. Additionally, the stem cells must overcome the hostile metabolic milieu in the ischemic brain, which includes poor blood flow, as well as the adverse metabolic environment that caused the stroke in the first place (eg. high blood sugar, high cholesterol, high blood pressure).

So in this proposal we are taking a different approach. We will develop methods to make blood vessels using human embryonic stem cells (HESC). HESC derived blood vessel cells will be injected into rats that have had a surgically-induced stroke. We will determine if the HESC-derived vascular cells find their way to the area of poor blood flow. We will determine if these cells survive and if they generate new blood vessels.

Furthermore, we plan to genetically engineer the HESCs to make them hardier. The area of stroke in the brain is a hostile environment for cells. One of the factors that mediates the adverse metabolic effects is a substance called ADMA (asymmetric dimethylarginine). We will engineer HESC that are more able to handle this substance, and determine if that genetic modification gives the HESC a better chance of surviving and forming blood vessels.

This proposal will provide insights into the use of human embryonic stem cells (HESC) for regenerating the injured brain after a stroke. These insights are likely to generate new intellectual property. The principal investigator has a track record of translational research. Our laboratory has generated fundamental biological insights that have been applied to disease models, and ultimately used to develop new therapies. The patents from our laboratory have had a high rate of licensure. From these patents there are now products in the pipeline, several in clinical trials, and one on the market. To summarize, support of this proposal is likely to generate fundamental new insights into the biology of vascular cells derived from human embryonic stem cells, and may lead to new therapeutic avenues that are desperately needed for stroke.

Review Summary:

SYNOPSIS: The PI suggests that hESC-derived endothelial cells (hESC-EC), after cerebrovascular ischemia may, a) restore a functional EC monolayer, reducing permeability, hemorrhage and inflammation, b) regenerate microvasculature, reducing apoptosis and c) establish a neurovascular niche, promoting neuronal repair. Infused hESC-EC must work despite the local presence of ADMA (asymmetric dimethylarginine), which competes with NO synthase for l-arginine and thus impairs endothelial and neuronal cell survival. Hence, a strategy is proposed to reduce the levels of ADMA. In this proposed study the investigators will look into the effectiveness of hESC-EC (both transgenic to overcome the effect of AMDA or wild-type) in the treatment of stroke (MCAO model in rats). The first aim is to generate a hESC cell line expressing a triple fusion reporter gene construct for bioluminescence, fluorescence and positron emission tomography (PET). Differentiation to EC from hESC will be achieved by an EC specific promoter VE-cadherin and the resultant EC will have the three different kinds of reporter genes: GFP (fluorescent), luciferase (bioluminescence) and HSV-tk (for PET). Whether EC derived from the transfected hESC line (expressing the construct) migrate from blood into the site of ischemia and mediate an effect will be determined. Accumulated EC will be delineated in vivo by either bioluminescent or PET imaging. The accumulated EC can be detected ex vivo by fluorescent microscopy or other immunohistochemistry techniques. The second aim is to determine and modulate the effects of ADMA on hESC-EC proliferation and incorporation into the vasculature.

SIGNIFICANCE AND INNOVATION: The clinical importance of reducing the neuropathologic consequences of stroke are obvious, and the proposed research represents an important step to utilize hESC-EC in the treatment of cerebrovascular ischemia or non-hemorrhagic stroke. Administration of endothelial cells at the site of lesions may prevent destructive sequences of stroke by active participation in repairing blood vessels or by releasing trophic factors for the survival of nascent EC, neurons or neural stem cells. The PI has raised the plausible hypothesis that transfused hESC-EC would be rapidly recruited into the area of ischemia and start the neurovascular recovery. The hope is that they would participate in the repair of the microvasculature, decreasing the inflammatory response and producing neurotrophic factors to improve the “neurovascular” milieu by releasing NO. The lessons to emerge may be useful beyond stroke; potentially, the properties that enable hESC-EC to traverse the blood-brain barrier in this case may allow other differentiated cells to do likewise.

STRENGTHS: The proposal is a pleasure to read because of its clarity and the directness of the rationale behind each experiment. One of the factors that inhibits the function of endothelial cells is methylated arginine analogues, most prominently asymmetric demethylarginine (AMDA), and this PI is an expert on ADMA and its relationship to endothelial function and angiogenesis. The PI also has been instrumental in developing the evidence for the beneficial effects of NO in the aftermath of cerebrovascular ischemia, and this proposal raises the possibility of modulating cellular ADMA by the administration of hESC-derived EC. A range of experiments, techniques and novel tools are proposed that are aimed at informing and documenting the position and status of the EC cells and their movements in vivo. In each case the PI has strong support from collaborator. Dr. Wu, an imaging expert for molecular imaging using bioluminescent and nuclear medicine techniques, provides a triply-reporting gene construct allowing both in vitro and in vivo monitoring of the transduced cells with high sensitivity. This construct should permit non-invasive detection and quantification of carrier cells undiminished by dilution or cell division. Similarly, the association with Dr. Steinberg and Dr. Giffard (a rodent MCAO model expert) contributes their experience with morphometric and immunohistologic evaluation of the post-ischemic brain.

The availability of animal models to study specific aspects of the EC interactions, including the mouse transgenic expressing DDAH (the enzyme that metabolizes ADMA) strengthens the proposal. In addition, the PI has access to numerous hESC lines, approved and non-approved, and the space needed for their proper handling.

WEAKNESSES: This key weakness of the proposal is that it lacks a high resolution anatomical imaging section. Although PET or bioluminescent imaging can delineate the accumulated transgenic EC (containing luciferase or HSV-tk gene), it cannot pinpoint the exact sites of accumulation. Localized, accumulated transgenic EC following multiple divisions may uptake enough luciferin or PET agent to give a false impression of the active incorporation of the administered ECs into angiogenesis. In addition multiple PET imaging may impair the function of the ECs and would deliver high radiation doses to rats.

In Research Design section 1.2b, the PI mentions that spectroscopy will be used to measure hemorrhage, but did not mention any protocol for either MRI or MRS. To significantly strengthen this proposal, the PI should add an MRI scanning protocol including MRS to detect the lesion size and the changes following administration of EC. Contrast enhanced images may be acquired to measure perfusion, permeability, CBV or CBF in the lesion and control hemispheres. Iron oxides can be used to label EC to delineate the early distribution of EC by cellular MRI. MRI can be performed repeatedly without impairing the function of administered EC or causing any adverse effect to the rats.

Another weakness of the proposal is the use of hESC-derived committed EC. These committed ECs may not be as effective as endothelial progenitor cells (EPC) to repair the injured vessels or to incorporate into active angiogenesis. In addition, while not a major weakness, it would be useful to have Dr. Cooke’s explanation of why hESC–derived EC cells are preferable to cells derived from adult sources.

DISCUSSION: Reviewers were enthusiastic about the experience of the PI, the strength of the assembled collaborators, the utility of the stroke model, and the clarity of the proposal. The studies would be performed smoothly and useful results would emerge. However, two major issues were highlighted by reviewers. First, the lack of high resolution MRI may result in the false impression that EC integrate into a site because of the luciferase reporter. Second, since endothelial progenitor cells can be obtained as peripheral blood progenitors, it seems that these could also work well in these studies. This alternative was not addressed.